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Human NK Cell Isolation Kit (Column-Free)

Human NK Cell Isolation Kit (Column-Free)

Catalog Number: S0K3001 Reactivity: Human Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Human NK Cell Biotin-Antibody Cocktail

2mL Streptavidin Beads
Capacity

Suitable for a total cell count of 1×10⁹, and can perform up to 100 sorting runs.
Principle of Separation

In this negative selection process, non-NK cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin‑coated nanomagnetic beads. As a result, the non‑target cells become bound to the magnetic beads via the antibody complexes, while the target NK cells remain unlabeled. Subsequently, the cell suspension is transferred to a polystyrene tube and placed within the magnetic field of the sorter. Under the influence of the magnetic field, the magnetically labeled non‑target cells adhere to the tube wall, whereas the unlabeled NK cells remain in the supernatant. This supernatant constitutes the NK cell‑enriched fraction after removal of the non‑target cells.
Reactivity Human
Magnetic Bead Size 150 nm
Separation Method Column-Free
Selection Strategy Negative Selection
Stability & Storage

Store protected from light at 2–8 °C; do not freeze.

Background

Human NK cells (Natural Killer Cells) are key effector cells of the innate immune system, accounting for 5%–20% of peripheral blood lymphocytes. They can directly kill virus-infected and tumor cells without prior antigen sensitization and secrete large amounts of cytokines to regulate immune responses, making them a crucial focus of research in tumor immunity, anti-infectious immunity, and immunotherapy. NK cells isolated using an NK cell negative selection kit are suitable for research scenarios where direct surface molecule labeling might interfere with downstream applications; the purified NK cells can be immediately used for flow cytometry, cell culture, or DNA/RNA extraction, among other downstream assays.

Protocol

Note: This kit does not include sorting buffer; please provide it separately. The corresponding Staret product number isS0D3018

Steps

Operating Instructions

Dosage and timing

1

Peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood by density-gradient centrifugation using lymphocyte separation medium, or cryopreserved PBMCs are directly thawed and resuspended, followed by cell counting.

Note: It is recommended to count cells after staining with AO/PI; PBMCs can be centrifuged at 400g for 7 minutes.

Prepare the sample

2

Resuspend 1×10 in 100 μL of MagSep Separation Buffer.sevencell;

Note:Reagents are adjusted proportionally based on the sample volume; a pre-sorting sample is retained for determining the proportion of human NK cells.

1×10sevencells/100 μL

3

Add 10 μL of the Human NK cell Biotin Antibody Cocktail to the sample and gently mix.

Note:In this step, gently pipette up and down 2–3 times to mix thoroughly;

10 μL/100 μL

4

Incubate the antibodies in the cocktail with the cells at room temperature for 10 minutes;

Incubate at room temperature for 10 minutes

5

Shake for 5–30 seconds to mix the Streptavidin beads;

Shake for 5–30 seconds

6

Add 20 μL of Streptavidin beads to the sample;

20 μL/100 μL

7

Gently mix the magnetic beads and cells, and incubate at room temperature for 5 minutes;

Note:For this step, you can use a pipette to gently blow the mixture 2–3 times to mix it evenly;

During this step, the magnetic beads may settle at the bottom; you can mix them again after about 2.5 minutes;

Incubate at room temperature for 5 minutes

8

Add MagSep Separation Buffer to the sample, and immediately place it on the magnetic stand to prevent the magnetic beads from settling heavily at the bottom;

Add the total volume to 2.5 mL (5 mL flow cytometry tube)

Add the total volume to 7.5 mL (15 mL centrifuge tube)

9

Place the sample on the magnetic stand so that the magnetic beads adhere to the tube wall;

Adsorb using a single-well magnetic stand (3 min)

Adsorb using a multi-well magnetic stand (5 min)

10

Tilt the magnetic stand and pour the sample into a new collection tube to collect the sample;

Note:The sorted cells can be centrifuged at 400g for 7 minutes, for subsequent culture and analysis.

Cell sorting successful

Note:For 0.1–1 × 10^8 cells (0.1–1 mL), it is recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) to adsorb a total volume of 2.5 mL;

2–5 × 10^8 cells (2–5 mL) are recommended to be adsorbed using a 15 mL row-type magnetic separator (Starter EasyEights EasyIso Separator), with a total volume of 7.5 mL;


Picture

Validation Data

Human NK cells were enriched from peripheral blood mononuclear cells (PBMCs). After fluorescent staining with CD56-APC (SDT Cat: S0B5623) and CD3-Alexa Fluor® 488 (SDT Cat: S0B1636), live cells (Live+) were gated for analysis. Flow cytometry results showed that the purity of the sorted human NK cells was 92.5%.